Project Summary Our studies integrate fundamental biochemical and biophysical basis of Ca2+ channel activity with animal models of human diseases. We seek an understanding of how native channels are constructed, activated and regulated, how they control important functions in physiological systems and how their altered function drives disease. We are focused on the Ca2+ signaling proteins, ORAI, STIM, MCU and NCLX. ORAI channels are encoded by three separate genes (Orai1-3) and activated by the endoplasmic reticulum (ER) Ca2+ sensing proteins STIM (STIM1- 2). ORAI represent a family of highly Ca2+ selective channels in the plasma membrane (PM) of virtually all cells. The founding member of this family, ORAI1 physically interacts with STIM1 in ER-PM junctions, to mediate store-operated Ca2+ entry (SOCE). SOCE is critical in controlling many physiological functions including secretion, migration and proliferation in virtually all cell types. However, the physiological roles of ORAI2, ORAI3 and the mammalian-specific translational variant of ORAI1 called ORAI1? remain poorly understood. ORAI-mediated Ca2+ signals propagate into mitochondria to regulate both bioenergetics, biogenesis and apoptosis. Mitochondrial Ca2+ uptake is decoded within the mitochondrial matrix, effectively coupling PM receptor activation to metabolic activity. Mitochondrial Ca2+ uptake and extrusion are mediated by the mitochondrial Ca2+ uniporter (MCU) and Na+/Ca2+ exchanger (NCLX), respectively. The mechanisms of reciprocal regulation between MCU/NCLX and STIM/ORAI and the convergence of these mechanisms in the control of metabolism, obesity and vascular disease are unknown. Results from our animal models and our compelling in vivo and in vitro data support specific roles for these Ca2+ signaling molecules at the ER-PM-mitochondria nexus, where they regulate cell signaling and metabolism of critical importance in endothelial dysfunction, obesity, hypertension and vascular remodeling. Our studies are aimed at understanding: 1) The fundamental mechanisms of organization, activation and regulation of these Ca2+ channels and their mechanisms of communication; and 2) The role of these Ca2+ signaling proteins in patho/physiology of vascular and metabolic disease. Our studies will provide novel insights into the precise role of these molecules in metabolic and signaling pathways controlling physiology and pathophysiology and will lead to novel avenues for disease therapy.